Protein-protein interactions regulate fundamental cellular events and misregulation of these interactions leads to disease states. Several approaches centered on high-throughput screening and rational design approaches are being evaluated for development of synthetic compounds as protein-protein interaction (PPI) inhibitors. Proteins often utilize small folded domains for recognition of other biomolecules. The basic hypothesis guiding our research is that by mimicking these folded domains we can specifically inhibit chosen PPIs with rationally designed synthetic molecules. Based on this hypothesis, we have developed cell permeable stabilized helices termed hydrogen bond surrogate or HBS helices to target intracellular PPIs. This work has created a foundation for the development of a new class of structure-based therapeutics. As part of these recent studies, we have also developed computational algorithms to predict targets for these helix mimics. The present proposal builds on this success to target therapeutically important interfaces that have not yielded to our past designs. We will build new experimental approaches to overcome hurdles. The three specific aims are to (1) develop a protein surface analysis approach to target recalcitrant helical and non-helical complexes, (2) establish an approach for target-guided assembly of stabilized helices, and (3) assess properties that aid cellular uptake of HBS helices and explore mechanism of entry for stabilized peptide helices. Studies in each Aim will make significant contributions to general approaches to inhibit PPIs and allow advancement of synthetic helices as distinct constructs spanning the molecular size space between small molecules and proteins.